Gene therapy offers hope of a cure for cystic fibrosis (CF) by correcting mutation of the cystic fibrosis transmembrane conductance regulator (CFTR). Though not yet a reality, the goals of this proposal are aimed at generating viral vectors that successfully target epithelial cells of the human airway. Presently, viral vectors are inefficient at infecting the airway epithelia due, in large part, to the absence of viral receptors at the apical surface. One approach to circumventing this limitation is to target viral vectors to receptors that are accessible from the apical surface. Identifying and characterizing these proteins is a first step towards utilizing them as targets for viral vectors. The urokinase plasminogen activator receptor (uPAR) is apically expressed by human airway epithelia. This proposal will further study uPAR's utility as a gene transfer-targeting molecule. Its expression in different epithelial cell types, association with potential co-receptors, ability to endocytose and mechanism of endocytosis will be examined. Additionally, a seven amino acid uPAR binding peptide (u7p) will be engineered into outer capsid proteins of two gene transfer vectors, adenovirus (Ad) and adeno-associated virus serotype 2 (AAV2). U7p-modified viral proteins will be analyzed for their ability to bind uPAR. Viral particles will then be tested on uPAR expressing cells. Moreover, another potential receptor system, erbB2 and 3, will be examined as potential targeting proteins. Both receptors are expressed by human airway epithelia. Their expression pattern, cell type specificity and ability to internalize will be analyzed for potential as targeting molecules for gene transfer vectors. Identifying other apically expressed proteins from human airway epithelia will generate more potential targeting molecules. A surface biotinylation approach will be used for their isolation. Proteins will be separated and identified by MALDI-MS. Of particular interest are those apical proteins that internalize because they allow for entry into the cell. The major goals of this proposal are to generate candidate-targeting molecules with the hope of increasing efficiency of gene transfer vectors to the airways.